Bottom Line:
In the current study the transcriptomics and physiological consequences of these morphological changes were investigated and compared with the data of the morphogenetic network model for the dichotomous branching mutant ramosa-1.The transcriptomic signatures and the reconstructed network model for all three morphology mutants (ΔracA, Rac(G18V), ramosa-1) imply that these pathways become integrated to bring about different physiological adaptations including changes in sterol, zinc and amino acid metabolism and changes in ion transport and protein trafficking.Finally, the fate of exocytotic (SncA) and endocytotic (AbpA, SlaB) markers in the dichotomous branching mutant ΔracA was followed, demonstrating that hyperbranching does not per se result in increased protein secretion.

ABSTRACTRacA is the main Rho GTPase in Aspergillus niger regulating polarity maintenance via controlling actin dynamics. Both deletion and dominant activation of RacA (Rac(G18V)) provoke an actin localization defect and thereby loss of polarized tip extension, resulting in frequent dichotomous branching in the ΔracA strain and an apolar growing phenotype for Rac(G18V). In the current study the transcriptomics and physiological consequences of these morphological changes were investigated and compared with the data of the morphogenetic network model for the dichotomous branching mutant ramosa-1. This integrated approach revealed that polar tip growth is most likely orchestrated by the concerted activities of phospholipid signaling, sphingolipid signaling, TORC2 signaling, calcium signaling and CWI signaling pathways. The transcriptomic signatures and the reconstructed network model for all three morphology mutants (ΔracA, Rac(G18V), ramosa-1) imply that these pathways become integrated to bring about different physiological adaptations including changes in sterol, zinc and amino acid metabolism and changes in ion transport and protein trafficking. Finally, the fate of exocytotic (SncA) and endocytotic (AbpA, SlaB) markers in the dichotomous branching mutant ΔracA was followed, demonstrating that hyperbranching does not per se result in increased protein secretion.

pone-0068946-g005: Biomass accumulation and morphology during submerged cultivation of PglaA-RacAG18V and PglaA-RacA mutant strains.(A) Growth curve for both mutant strains. The dashed line indicates the time point when the inducing carbon source maltose was added. The two arrows indicate the time points at which samples for transcriptome analysis were taken. (B) Dispersed hyphal morphology at the time point of maltose addition as well as 2 and 4 h after maltose addition. Bar, 20 µm.

Mentions:
Next, we wished to dissect the transcriptomic adaptation of A. niger to dominant activation of RacA. Batch cultures of our previously established RacA mutant strains PglaA::racAG18V and PglaA::racA (reference strain) were started with xylose (0.75%) as repressing carbon source. After the cultures reached the exponential growth phase and xylose was consumed, maltose (0.75%) was added to induce expression of genes under control of PglaA. Hypothetically, a so far unknown RacA-dependent GAP ensures that the activity of RacA is spatially restricted to the hyphal apex in the PglaA-racA strain thereby maintaining a stable polarity axes even under inducing conditions (Fig. 5). However, this control mechanism is leveraged off in the PglaA-racAG18V mutant strain, as the GTPase-negative G18V mutation traps RacA in its active, GTP-bound form [24]. Hence, the switch from xylose to maltose leads to a loss of polarity maintenance in the PglaA-racAG18V strain and the formation of clavate-shaped hyphal tips and bulbous lateral branches (Fig. 5). RNA samples were extracted from duplicate cultures 2 and 4 h after the maltose shift and used for transcriptomic comparison. Expression of 3,757 (506) genes was modulated after 2 h (4 h) of induction, 1,906 (282) of which showed increased and 1,851 (224) decreased expression levels in the PglaA-racAG18V strain when compared to the PglaA-racA reference strain (FDR<0.05). The complete list of differentially expressed genes, including fold change and statistical significance is given in Tables S1 and S2. GO enrichment analysis using the FetGOat tool [43] discovered that most of these genes belong to primary metabolism, suggesting that both strains differed in their ability to quickly adapt to the new carbon source (Table S3).

pone-0068946-g005: Biomass accumulation and morphology during submerged cultivation of PglaA-RacAG18V and PglaA-RacA mutant strains.(A) Growth curve for both mutant strains. The dashed line indicates the time point when the inducing carbon source maltose was added. The two arrows indicate the time points at which samples for transcriptome analysis were taken. (B) Dispersed hyphal morphology at the time point of maltose addition as well as 2 and 4 h after maltose addition. Bar, 20 µm.

Mentions:
Next, we wished to dissect the transcriptomic adaptation of A. niger to dominant activation of RacA. Batch cultures of our previously established RacA mutant strains PglaA::racAG18V and PglaA::racA (reference strain) were started with xylose (0.75%) as repressing carbon source. After the cultures reached the exponential growth phase and xylose was consumed, maltose (0.75%) was added to induce expression of genes under control of PglaA. Hypothetically, a so far unknown RacA-dependent GAP ensures that the activity of RacA is spatially restricted to the hyphal apex in the PglaA-racA strain thereby maintaining a stable polarity axes even under inducing conditions (Fig. 5). However, this control mechanism is leveraged off in the PglaA-racAG18V mutant strain, as the GTPase-negative G18V mutation traps RacA in its active, GTP-bound form [24]. Hence, the switch from xylose to maltose leads to a loss of polarity maintenance in the PglaA-racAG18V strain and the formation of clavate-shaped hyphal tips and bulbous lateral branches (Fig. 5). RNA samples were extracted from duplicate cultures 2 and 4 h after the maltose shift and used for transcriptomic comparison. Expression of 3,757 (506) genes was modulated after 2 h (4 h) of induction, 1,906 (282) of which showed increased and 1,851 (224) decreased expression levels in the PglaA-racAG18V strain when compared to the PglaA-racA reference strain (FDR<0.05). The complete list of differentially expressed genes, including fold change and statistical significance is given in Tables S1 and S2. GO enrichment analysis using the FetGOat tool [43] discovered that most of these genes belong to primary metabolism, suggesting that both strains differed in their ability to quickly adapt to the new carbon source (Table S3).

Bottom Line:
In the current study the transcriptomics and physiological consequences of these morphological changes were investigated and compared with the data of the morphogenetic network model for the dichotomous branching mutant ramosa-1.The transcriptomic signatures and the reconstructed network model for all three morphology mutants (ΔracA, Rac(G18V), ramosa-1) imply that these pathways become integrated to bring about different physiological adaptations including changes in sterol, zinc and amino acid metabolism and changes in ion transport and protein trafficking.Finally, the fate of exocytotic (SncA) and endocytotic (AbpA, SlaB) markers in the dichotomous branching mutant ΔracA was followed, demonstrating that hyperbranching does not per se result in increased protein secretion.

ABSTRACTRacA is the main Rho GTPase in Aspergillus niger regulating polarity maintenance via controlling actin dynamics. Both deletion and dominant activation of RacA (Rac(G18V)) provoke an actin localization defect and thereby loss of polarized tip extension, resulting in frequent dichotomous branching in the ΔracA strain and an apolar growing phenotype for Rac(G18V). In the current study the transcriptomics and physiological consequences of these morphological changes were investigated and compared with the data of the morphogenetic network model for the dichotomous branching mutant ramosa-1. This integrated approach revealed that polar tip growth is most likely orchestrated by the concerted activities of phospholipid signaling, sphingolipid signaling, TORC2 signaling, calcium signaling and CWI signaling pathways. The transcriptomic signatures and the reconstructed network model for all three morphology mutants (ΔracA, Rac(G18V), ramosa-1) imply that these pathways become integrated to bring about different physiological adaptations including changes in sterol, zinc and amino acid metabolism and changes in ion transport and protein trafficking. Finally, the fate of exocytotic (SncA) and endocytotic (AbpA, SlaB) markers in the dichotomous branching mutant ΔracA was followed, demonstrating that hyperbranching does not per se result in increased protein secretion.